Summary Homologous recombination (HR) is essential for the maintenance of genome integrity. Rad51 paralogs fulfill a conserved, but undefined role in HR, and their mutations are associated with increased cancer risk in humans. Here, we use single–molecule imaging to reveal that the Saccharomyces cerevisiae Rad51 paralog complex Rad55–Rad57 promotes the assembly of Rad51 recombinase filaments through transient interactions, providing evidence that it acts as a classical molecular chaperone. Srs2 is an ATP–dependent anti–recombinase that downregulates HR by actively dismantling Rad51 filaments. Contrary to the current model, we find that Rad55– Rad57 does not physically block the movement of Srs2. Instead, Rad55–Rad57 promotes rapid re– assembly of Rad51 filaments after their disruption by Srs2. Our findings support a model in which Rad51 is in flux between free and ssDNA–bound states, the rate of which is dynamically controlled though the opposing actions of Rad55–Rad57 and Srs2.
Several important anti-tumor agents form DNA interstrand crosslinks (ICLs), but their clinical efficiency is counteracted by multiple complex DNA repair pathways. All of these pathways require unhooking of the ICL from one strand of a DNA duplex by nucleases, followed by bypass of the unhooked ICL by translesion synthesis (TLS) polymerases. The structures of the unhooked ICLs remain unknown, yet the position of incisions and processing of the unhooked ICLs significantly influence the efficiency and fidelity of bypass by TLS polymerases. We have synthesized a panel of model unhooked nitrogen mustard ICLs to systematically investigate how the state of an unhooked ICL affects pol η activity. We find that duplex distortion induced by a crosslink plays a crucial role in translesion synthesis, and length of the duplex surrounding an unhooked ICL critically affects polymerase efficiency. We report the synthesis of a putative ICL repair intermediate that mimics the complete processing of an unhooked ICL to a single crosslinked nucleotide, and find that it provides only a minimal obstacle for DNA polymerases. Our results raise the possibility that, depending on the structure and extent of processing of an ICL, its bypass may not absolutely require TLS polymerases.
Abstract DNA polymerase ζ (Pol ζ) and Rev1 are essential for the repair of DNA interstrand crosslink (ICL) damage. We have used yeast DNA polymerases η, ζ, and Rev1 to study translesion synthesis (TLS) past a nitrogen mustard-based ICL with an 8-atom linker between the crosslinked bases. The Rev1-Pol ζ complex was most efficient in complete bypass synthesis, by 2-3 fold, compared to Pol ζ alone or Pol η. Rev1 protein, but not its catalytic activity, was required for efficient TLS. A dCMP residue was faithfully inserted across the ICL-G by Pol η, Pol ζ, and Rev1-Pol ζ. Rev1-Pol ζ, and particularly Pol ζ alone showed a tendency to stall before the ICL, whereas Pol η stalled just after insertion across the ICL. The stalling of Pol η directly past the ICL is attributed to its autoinhibitory activity, caused by elongation of the short ICL-unhooked oligonucleotide (a six-mer in our study) by Pol η providing a barrier to further elongation of the correct primer. No stalling by Rev1-Pol ζ directly past the ICL was observed, suggesting that the proposed function of Pol ζ as an extender DNA polymerase is also required for ICL repair.
DNA interstrand crosslinks (ICLs) that are repaired in non-dividing cells must be recognized independently of replication-associated DNA unwinding. Using cell-free extracts from Xenopus eggs that support neither replication nor transcription, we establish that ICLs are recognized and processed by the mismatch repair (MMR) machinery. We find that ICL repair requires MutSα (MSH2-MSH6) and the mismatch recognition FXE motif in MSH6, strongly suggesting that MutSα functions as an ICL sensor. MutSα recruits MutLα and EXO1 to ICL lesions, and the catalytic activity of both these nucleases is essential for ICL repair. As anticipated for a DNA unwinding-independent recognition process, we demonstrate that least distorting ICLs fail to be recognized and repaired by the MMR machinery. This establishes that ICL structure is a critical determinant of repair efficiency outside of DNA replication.
